The invention provides a novel vaccine adjuvant comprising lipopolysaccharide (LPS) antagonist, and use of the same in vaccine preparations and methods of vaccinating a subject comprising a vaccine antigen and a pharmaceutically active amount of an LPS antagonist.
An adjuvant is a compound that, when combined with a vaccine antigen, increases the immune response to the vaccine antigen over that induced by the vaccine antigen alone. Strategies that promote antigen immunogenicity include those that render vaccine antigens particulate, polymerize vaccine antigens, emulsify vaccine antigens, encapsulate vaccine antigens, increase host innate cytokine responses, or target vaccine antigens to antigen presenting cells (Nossal, 1999, In: Fundamental Immunology. Paul (Ed.), Lippincott-Raven Publishers, Philadelphia, Pa.; Vogel and Powell, 1995, In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p. 141). Because of the essential role adjuvants play in improving the immunogenicity of vaccine antigens, the use of adjuvants in the formulation of vaccines has been virtually ubiquitous (Nossal, 1999, supra; Vogel and Powell, 1995, supra; see also PCT publication WO 97/18837, the teachings of which are incorporated herein by reference).
A compendium of adjuvants with know immunomodulatory properties is available (Vogel and Powell, 1995, supra). Examples of well-known adjuvants include Freund""s adjuvant (Vogel and Powell, 1995, supra), MF59 (Vogel and Powell, 1995, supra; Ott, et al., 1995, In; Vaccine Design. The subunit and adjuvant approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p.277; Traquina, et al., 1996, J. Infect. Dis., 174:1168; Ott, et al., 1995, Pharm. Biotechnol., 6:277), SAF-1 (Vogel and Powell, 1995, supra), polylactide co-glycolide encapsulation (PLG; (Vogel and Powell, 1995, supra; Eldridge, et al., 1991, Infect. Immun., 59:2978; Eldridge, et al., 1993, Semin. Hematol., 30:16; Vordermeier, et al., 1995, Vaccine 13:1576; Ugozzoli, et al., 1998, Immunol., 93:563), aluminium hydroxide/phosphate (xe2x80x9cAlumxe2x80x9d; (Vogel and Powell, 1995, supra; Edelman, 1980, Rev. Infect. Dis., 2:370; Seppala and Makela, 1984, Immunol., 53:827; Shirodkar, et al., 1990, Pharm Res., 7:1282; Weissburg, et al., 1995, Pharm. Res., 12:1439), and immune-stimulating complexes (xe2x80x9cISCOMsxe2x80x9d; Vogel and Powell, 1995, supra; Rimmelzwaan and Osterhaus, 1995, In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p.543; Sjolander, et al., 1998, J. Leukoc. Biol., 64:713; Morein, 1990, Vet. Microbiol., 23:79; Lovgren and Morein, 1991, Molec. Immunol., 28:285).
A more recent adjuvant strategy has been to use purified recombinant cytokines as adjuvants (Vogel and Powell, 1995, supra). Cytokines that have been employed in this fashion include IL-1a, TNFa, IL-2, IL4, IL-6, IL-12, interferon-gamma (IFN-xcex3), and granulocyte monocyte colony stimulating factor (GM-CSF; Nossal, 1999, supra; Vogel and Powell, 1995, supra; Nash, et al., 1993, Immunol. Cell. Biol., 71:367; Pardoll, 1995, Annu. Rev. Immunol., 13:399; Kurane, et al., 1997, Ann. Surg. Oncol., 4:579; Tagliabue and Boraschi, 1993, Vaccine, 11:594; Lofthouse, et al., 1995, Vaccine, 13:1131; Pasquini, et al., 1997, Immunol. Cell. Biol., 75:397; Jankovic, et al., 1997, J. Immunol., 159:2409).
The advent of nucleic acid vaccines has enabled new avenues in the field of adjuvant development. This includes the use of nucleic acid vaccines that encode cytokines, such as IL-4, IFN-xcex3 and GM-CSF, in addition to the vaccine antigen (Pasquini, et al., 1997, supra) and nucleic acid vaccines that encode co-stimulatory molecules such as CD80 (also known as B7.1), in addition to the vaccine antigen (Pasquini, et al., 1997, supra).
Although there is no single mechanism of adjuvant action, an essential characteristic of adjuvants is their ability to significantly increase the level of immunity to a vaccine antigen over the level of immunity induced by the vaccine antigen alone (Nossal, 1999, supra; Vogel and Powell, 1995, supra). In this regard, some adjuvants are more effective at augmenting humoral immune responses; other adjuvants are more effective at increasing cell-mediated immune responses (Vogel and Powell, 1995, supra); and yet another group of adjuvants increase both humoral and cell-mediated immune responses against vaccine antigens (Vogel and Powell, 1995, supra).
Several adjuvants are derived from bacterial products. Bacteria are composed of a diverse array of biologically active components that have proven adjuvant activity, including porins, cholera toxin (also called xe2x80x9cCxe2x80x9d), heat-labile toxin of enterotoxigenic E. coli (also called LT), muramyl dipeptide, lipoarabinomannans (xe2x80x9cLAMxe2x80x9d), lipid A, and monophosphoryl-lipid A (Nossal, 1999, supra; Vogel and Powell, 1995, supra; Lidgate and Byars, 1995, In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p.313; Ulrich and Myers, 1995, In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p.495). In addition, unmethylated CpG DNA sequences, which are expressed by bacteria or made synthetically, have been shown to possess potent immunostimulatory properties (Davis, et al., 1998, J. Immunol., 160:870; Klinman, 1998, Antisense Nucleic Acid Drug Dev., 8:181; Liu, et al., 1998, Blood, 92:3730; Wloch, et al., 1998, Hum. Gene. Therap., 9:1439; Brazolot-Milla, et al., 1998, Proc. Natl. Acad. Sci. USA, 95:15553; Moldoveanu, et al., 1998, Vaccine, 16:1216). Inclusion of such sequences in nucleic acid vaccines is thought to play a key role in the immunogenicity of DNA vaccines (Davis, et al., 1998, supra; Klinman, 1998, supra; Liu, et al., 1998, supra; Wloch, et al., 1998, supra; Brazolot Milla, et al., 1998, supra; Moldoveanu, et al., 1998, supra).
LPS (lipopolysaccharide), also referred to as xe2x80x9cendotoxinxe2x80x9d, is the major surface component of gram negative bacteria. Under normal conditions, LPS is inserted in the outer surface of the outer membrane of gram negative bacteria (Schnaitman and Klena, 1993, Microbiol. Rev., 57:655; Makela and Stocker, 1984, In: Handbook of Endotoxin volume 1, Elsevier Biomedical Press, Amsterdam, Rietschel (Ed.), pp. 59-137). Complete or xe2x80x9csmoothxe2x80x9d LPS is composed of three main domains called lipid A, the O-antigen (also called the O-polysaccharide) and the core region, which creates an oligosaccharide link between lipid A and the O antigen (Schnaitman and Klena, 1993, supra; and Makela and Stocker, 1984, supra). The O-antigen is composed of oligosaccharide repeat units. The structure and number of these repeats varies depending on the bacterial species and growth conditions, typically ranging from one to fifty repeats (Schnaitman and KIena, 1993, supra; and Makela and Stocker, 1984, supra). Some bacterial generi, such as Neisseria spp., produce LPS that has low numbers of O-antigen repeats and therefore is referred to as lipooligosaccharide (LOS) simply to reflect this fact (Schnaitman and Klena, 1993, supra; and Makela and Stocker, 1984, supra).
The biologically active component of LPS is lipid A (Rietschel, et al., 1994, FASEB J., 8:217; Verma, et al., 1992, Infect. Immun., 60:2438; Alving, 1991, J. Immunol. Meth., 140:1; Alving and Richards, 1990, Immunol. Lett., 25:275; Richard, et al., 1988, Infect. Immun., 56:682. Activity analysis of lipid A biosynthesis precursors or synthetic intermediates showed that various elements of lipid A are essential for pyrogenicity (Rietschel, et al., 1994, supra; Raetz, et al., 1985, J Biol. Chem., 260:16080). Lipid X and lipid IVa are completely non pyrogenic precursor forms of lipid A (Wang, et al., 1991, Infect. Immun., 59:4655; Ulmer, et al., 1992, Infect. Immun., 60:145; Kovach, et al., 1990, J. Exp. Med., 172:77).
Lipid X is a monosaccharide precursor of lipid A (Rietschel, et al., 1994, supra). Lipid IVa, a tetraacyl precursor of lipid A, is interesting in that it retains the ability to bind to host cell surfaces but has no pyrogenicity, suggesting that binding to host cell surfaces per se does not impart this biological property (Wang, et al., 1991, supra; Ulmer, et al., 1992, supra; Kovach, et al., 1990 supra).
The genetics of lipid A biosynthesis are well described (e.g., Raetz, et al., 1985, supr,. Raetz, 1990, Ann. Rev. Biochem., 59:129). The majority of mutations that prevent the biosynthesis of lipid A, such as mutations in the IpxA, lpxB, kdsA, kdsB, and kdtA genes, are lethal as the biosynthesis of lipid A is essential for cell survival (Rick, et al., 1977, J. Biol. Chem., 252:4904; Rick and Osborne, 1977, J. Biol. Chem., 252:4895; Raetz, et al., 1985, supra; Raetz, et al., 1990, supra; Raetz, et al., 1993, supra; Schnaitman and Klena, 1993, supra). For the most part, therefore, analysis of these genes has involved the use of temperature-sensitive mutants, which only display null phenotypes under non-permissive conditions (Rick, et al., 1977, supra; Rick and Osborne, 1977, supra; Raetz, et al., 1985, supra; Raetz, et al., 1990, supra; Raetz, et al., 1993, supra; Schnaitman and Klena, 1993, supra). When grown under non permissive conditions, lpxA, kdsA, kdsB, and kdtA mutants accumulate non-pyrogenic precursor forms of LPS (to about 50% of the total LPS), such as lipid X or lipid IVa.
There is now evidence that mutations in htrB and msb may influence the biosynthesis of lipid A (Karow, et al., 1991, J. Bacteriol., 173:741; Karow and Georgopoulos, 1992, J. Bacteriol., 174:702). These mutants are temperature sensitive and LPS isolated from these mutants stains less intensely on silver-stain gels (Karow and Georgopoulos, 1992, supra). The basis for the temperature-sensitive growth phenotype of the htrB and msb mutants has remained cryptic (Karow and Georgopoulos, 1992, supra). There was speculation that these mutants produce defective lipid A precursors (Karow and Georgopoulos, 1992, supra). This was based on the observation that ammonium cationic compounds enabled these mutants to grow in non permissive temperatures (Karow and Georgopoulos, 1992, supra). These investigators proposed that the ammonium cationic compounds influenced the intermolecular interaction between LPS molecules in the outer membrane. This observation is supported by a report showing that an htrB mutant of Haemophilus influenzae produces modified LOS structures (Lee, et al., 1995, Infect. Immun., 63:818; Lee, et al., 1995, In: Abstracts of the American Society for Microbiology, ASM Washington D.C., p.206 (B-234)). Later studies showed direct evidence that htrB and msb mutants could produce substantially pure non-pyrogenic LPS (see PCT International Publication Nos. WO 97/18837 and WO 99/15162, the teachings of which are incorporated herein by reference).
Other lipid A precursor structures isolated from E. coli mutants that are defective in the biosynthesis of lipid A are described in Raetz, et al., 1985, supra; Kovach, et al., 1990, J. Exp. Med., 172:77; Golenbock, et al., 1991, J. Biol. Chem., 266:19490; Golenbock, et al., 1988, Antimicrob. Agents Chemother., 32:37; Clementz, et al., 1996, J. Biol. Chem., 271:12095; Clementz, et al., 1997, J. Biol. Chem., 272:10353; Garrett, et al., 1998, J. Biol. Chem., 273:12457; Kitchens, et al., 1992, J. Exp. Med., 176:485; Kitchens, and Munford, 1995, J. Biol. Chem., 270:9904; Munford and Hunter, 1992, J. Biol. Chem., 267:10116; Rietschel, et al., 1994, supra; Ulmer, et al., 1992, Infect. Immun., 60:5145; Ulmer, et al., 1992, Infect. Immun., 60:3309; Wang, et al., 1990, FEMS Micro. Immunol., 2:179; Wang, et al., 1991, Infect. Immun., 59:4655 64.
Synthetic lipid A partial structures are described in Wang, et al., 1990, FEMS Microbiol. Immunol., 2:179; Golenbock, et al., 1991, J. Biol. Chem., 266:19490; Ulmer, et al., 1992, Infect. Immun., 60:3309; Ulmer, et al., 1992, Infect. Immun., 60:5145; Perera, et al., 1993, Infect. Immun., 61:2015; Rietschel, et al., 1994, supra. Rhodobacter sphaeroides naturally produces an unusual diphosphoryl-lipid A structure (hereafter referred to as RsDPLA) that lacks pyrogenic activity (Qureshi, et al., 1991, J. Biol. Chem., 266:6532; Qureshi, et al., 1991, Infect. Immun., 59:441; Zuckerman and Qureshi, 1992, Infect. Immun., 60:2581; Kirikae, et al., 1995, Infect. Immun., 63:486; Qureshi, et al., 1997, J. Biol. Chem., 272:10594).
Through these studies a number of lipid A partial structures have been identified that lack pyrogenic activity and are LPS antagonists (i.e., they effectively block the pyrogenic activity of LPS from gram negative bacteria, e.g., E. coli LPS) including lipid X, tetraacyl-lipid A (LA4), pentaacyl-lipid A (LAS), and RsDPLA (Kovach, et al., 1990, supra; Golenbock, et al., 1991, supra; Golenbock, et al., 1988, supra; Kitchens, et al., 1992, supra; Kitchens and Munford, 1995, supra; Munford and Hunter, 1992, supra; Rietschel, et al., 1994, supra; Ulmer, et al., 1992, supra; Ulmer, et al., 1992, supra; Wang, et al., 1990, supra; Wang, et al., 1991, supra; Qureshi, et al., 1991, supra; Qureshi, et al., 1991, supra; Zuckerman and Qureshi, 1992, supra; Kirikae, et al., 1995, supra; Qureshi, et al., 1997, supra).
More recently, E. coli msbB mutants have been shown to produce LAS and LPS preparations isolated from E. coli msbB mutants were shown to possess LPS antagonist activity (Clementz, et al., 1997, supra; Somerville, et al., 1996, J. Clin. Invest., 97:359). Similarly, other investigators have shown that E. coli hirB and msbB mutants (Clementz, et al., 1996, supra; Clementz, et al., 1997, supra; Hone, et al., 1998, J. Human Virol., 1:251), Salmonella htrB mutants (Sunshine, et al., 1997, J. Bacteriol., 179:5521; Jones, et al., 1997, Infect. Immun., 65:4778), and msbB mutants (Low, et al., 1999, Nature Biotech., 17:37; Khan, et al., 1998, Mol. Microbiol., 29:571), and Haemophilus htrB mutants (Lee, et al., 1995, J. Biol. Chem., 270:27151) produce defective lipid A structures, predominated by LA4 and LA5, that are non pyrogenic and display LPS antagonist activity.
These findings also provided compelling evidence that the products of the htrB and msbB genes play a central role in the final steps of lipid A biosynthesis in gram negative bacteria that produce LAS or hexaacyl-lipid A structures, such as Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., and Vibrio spp. (Clementz, et al., 1997, supra; Somerville, et al., 1996, supra; Lee, et al., 1995, supra; Low, et al., 1999, supra; Khan, et al., 1998, supra; Sunshine, et al., 1997, supra; Jones, et al., 1997, supra).
Despite the diverse number of lipid A structures that have been identified that are non pyrogenic and display LPS antagonist activity, a consistent finding that emerged from the above described research effort was that lipid A precursors and partial structures that possess LPS antagonist activity (i.e., those produced by E. coli mutants and those made synthetically) failed to reproducibly display an effective adjuvant activity (Rietschel, et al., 1994, supra; Kotani, et al., 1986, Infect. Immun., 54:673; Hamann, et al., 1998, Int. J. Food Microbiol., 41:141; Loppnow, et al., 1990, Adv. Exp. Med. Biol., 256:561). In one instance the putative immunostimulatory activity of the LPS antagonist lipid X was subsequently shown to be due to the presence of a contaminant in the preparation. Once the contaminant was completely eliminated the immunostimulatory activity was completely lost (Lam, et al., 1991, Infect. Immun., 59:2351). Other reports claiming immunostimulatory activity in LPS antagonists are now thought to be due to contaminating protein in the LPS antagonist preparations (Hogan and Vogel, 1987, J. Immunol., 139:3697; Hogan and Vogel, 1988, J. Immunol., 141:4196).
Notwithstanding the above disappointing revelation, some lipid A structures that display markedly reduced pyrogenicity, were shown to retain adjuvant activities; a well known example in this category is monophosphoryl-lipid A (referred to herein as MPLA; Saha, et al., 1997, Immunopharmacol., 37:175-84; Hagen, et al., 1997, J Chromatogr., 767:53; Ulrich and Myers, 1995, Pharm. Biotechnol., 6:495; Zhou and Huang, L., 1993, Vaccine, 11:1139; Tabatabai, et al., 1992, Am. J. Vet. Res., 53:1900; Schneerson, et al., 1991, J. Immunol., 147:2136; Masihi, et al., 1986, Int. J. Immunopharmacol., 8:339; Johnson and Tomai, 1990, Adv. Exp. Med. Biol., 256:567-79). However, while MPLA displays substantially reduced pyrogenic activity, it induces modest levels of pyrogenic cytokines and does not possess LPS antagonist activity (Saha, et al., 1997, supra; Hagen, et al., 1997, supra; Ulrich and Myers, 1995, supra; Zhou and Huang, 1993, supra; Tabatabai, et al., 1992, supra; Schneerson, et al., 1991, supra; Masihi, et al., 1986, Int. J. Immunopharmacol. 8:339; Johnson and Tomai, 1990, supra).
Accordingly, the prior art teaches us that LPS antagonists, when used as highly purified preparations, display poor adjuvant properties compared to other widely used adjuvants such as those described in Section 2 above. That is, the prior art does not provide LPS, lipid A or derivatives thereof that are effective LPS antagonists and adjuvants, and lack pyrogenic activity.
The invention relates to methods and compositions comprising an adjuvant which is both an LPS antagonist and is non-pyrogenic. In a preferred embodiment, the non-pyrogenic LPS antagonist adjuvant is isolated from a gram negative bacterial strain that contains at least one mutation in at least one of the htrB and msbB genes.
In one aspect, the invention features an adjuvant comprising an LPS antagonist, wherein said LPS antagonist is isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes. In one embodiment, the LPS antagonist has reduced pyrogenicity. In a preferred embodiment, the LPS antagonist has substantially reduced pyrogenicity. In a more preferred embodiment, the LPS antagonist in non-pyrogenic. In a preferred embodiment, pyrogenicity is determined by measuring the levels of indicators of pyrogenicity or inflammation, such as, for example, IL-1xcex2, IL-6 or TNFxcex1, in a cell, extracellular medium, or a subject. In a most preferred embodiment, the LPS antagonist elicits no detectable TNFxcex1 activity when contacted with a cell or administered to a subject.
The LPS antagonist may comprise an LPS derivative or a fragment thereof (e.g., a precursor component or derivative thereof), such as a lipid A precursor structure selected from the group consisting of lipid X, tetraacyl-lipid A (LA4), pentaacyl-lipid A (LAS), or a Rhodobacter sphaeroides diphosphoryl-lipid A structure (RsDPLA). The LPS antagonist can be purified from a gram negative bacteria is selected from the group consisting of Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., and Vibrio spp.
In another embodiment, the gram negative bacterium defective in at least one of the msbB or htrB genes is also defective in at least one of the kdsA, kdsB, kdtA, lpxA, lpxB, lpxC lpxD or ssc genes.
In another embodiment, the invention provides a pharmaceutical preparation comprising a vaccine antigen, a pharmaceutically effective amount of an LPS antagonist, isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes, and a pharmaceutically acceptable carrier. The vaccine antigen may be any vaccine antigen, such as e.g., a polysaccharide, a protein or a nucleic acid. In an embodiment of the invention, the vaccine antigen is derived from a viral pathogen selected from the group consisting of orthomyxoviruses, retroviruses, herpesviruses, lentiviruses, rhabdoviruses, picornoviruses, poxviruses, rotavirus and parvoviruses. Exemplary antigens are influenza virus, RSV, EBV, CMV, herpes simplex virus, human immunodeficiency virus, rabies, poliovirus and vaccinia, human immunodeficiency virus antigens Nef, p24, gp120, gp41, Tat, Rev, and Pol; T cell and B cell epitopes of gp120; the hepatitis B surface antigen; rotavirus antigens, such as VP4 and VP7; influenza virus antigens such as hemagglutinin or nucleoprotein; and herpes simplex virus thymidine kinase.
In another embodiment the vaccine is derived from a bacterial pathogen selected from the group consisting of Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp., and Borellia burgdorferi. Exemplary vaccine antigens useful in the practice of the invention are the capsular polysaccharide of Neisseria meningitis; the Vi polysaccharide of Salmonella enterica serovar typhi; Shigella sonnei form 1 antigen; the O-antigen of V. cholerae Inaba strain 569; cholera toxin of V. cholerae; TCP of V. cholera; CFA/I fimbrial antigen of enterotoxigenic E. coli; the heat-labile toxin of E. coli; pertactin of Bordetella pertussis; adenylate cyclase-hemolysin of B. pertussis, and fragment C of tetanus toxin of Clostridium tetani. 
In another embodiment of the invention the vaccine antigen is derived from a parasitic pathogen selected from the group consisting of Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eimeria spp., Leishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp. Exemplary parasitic vaccine antigen useful in the practice of the invention are the circumsporozoite antigen of P. berghei, the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp.; the galactose specific lectin of Entamoeba histolytica; gp63 of Leishmania spp.; paramyosin of Brugia malayi; the triose-phosphate isomerase of Schistosoma mansoni; the secreted globin-like protein of Trichostrongylus colubriformis; the glutathione-S-transferase of Frasciola hepatica, Schistosoma bovis and S. japonicum; and KLH of Schistosoma bovis. 
In additional embodiments of the invention the vaccine antigen is derived from a tumor antigen selected from the group consisting of prostate specific antigen, TAG-72, carcinoembrionic antigen (CEA), MAGE-1, tyrosinase, and mutant p53 antigen; the CD3 receptor on T cells; an autoimmune antigen; or the IAS xcex2 chain. Alternatively, the invention can be practiced with a vaccine antigen such as an immuno-stimulatory molecule selected from the group consisting of M-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IFN-xcex3.
An another aspect, the invention features combination therapies, comprising a pharmaceutical composition containing the LPS antagonist together with other agents that enhance the activity of the LPS antagonist (e.g., by increasing its adjuvant or antagonist activities or promoting stabilization of the LPS antagonist or other components of the pharmaceutical composition) or minimize deleterious effects of the LPS antagonist.
In another aspect, the invention features methods for preparing and using an adjuvant as described herein comprising an LPS antagonist, wherein said LPS antagonist is isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes.
In another aspect, the invention features methods for preparing and using a vaccine comprising a vaccine antigen and a pharmaceutically effective amount of an LPS antagonist isolated from a gram negative bacterium that is defective in at least one of the msbB or htrB genes, and a pharmaceutically acceptable carrier.